The objective of this research proposal is to advance the molecular understanding of the basic process of protein transport across biological membranes. Our particular focus is on the signal recognition particle (SRP) as it plays a central role in protein secretion. The SRP RNA is an essential component of the SRP and contains several phylogenetically conserved sites of functional importance. The goal of our research is to understand the structural and functional role of this RNA in the human system. We will define those regions of the human SRP RNA which are involved in particular aspects of SRP-mediated protein secretion and of SRP assembly by using a combination of site-directed mutagenesis, site-directed cross-linking, structure probing, and phylogenetic comparisons with cloned SRP-components. The major topics to be investigated are: (1) The molecular mechanisms that involve SRP RNA in signal recognition and protein translocation. Human SRP will be reconstituted entirely from recombinant components that will be altered by site-directed mutagenesis to test the translocation competence of secretory proteins. Individual events in the SRP-cycle, such as signal recognition, translation arrest, and release of the arrest, will be identified. An RNA switch in the large SRP domain which controls the binding of protein SRP54 will be characterized. (2) Assembly of the SRP. Purified recombinant SRP components will be used to determine in detail the events in the SRP assembly pathway. The interactions of the SRP proteins with the SRP RNA will be studied by systematic site-directed mutagenesis, and RNA-protein neighborhoods will be determined by using site-directed photochemical cross-linking followed by the identification of the cross-linked nucleotides and amino acids. The data from these experiments will be viewed collectively to refine a three-dimensional model of the SRP RNA. (3) Biophysical characterization of the domain of protein SRP54 that associates closely with the signal peptide and interacts with SRP RNA. These studies will focus on the specific features of a critical RNA-protein interaction in the most conserved region of the SRP RNA and will define its relationship to the overall process of protein translocation.